U.S. patent number 4,352,557 [Application Number 06/141,638] was granted by the patent office on 1982-10-05 for determining a test value corresponding to blood subsidence.
This patent grant is currently assigned to Ernst Leitz Wetzlar GmbH. Invention is credited to Holger Kiesewetter, Klaus Mussler, Heinz Myrenne, Andreas Scheffler, Holger Schmid-Schonbein.
United States Patent |
4,352,557 |
Schmid-Schonbein , et
al. |
October 5, 1982 |
Determining a test value corresponding to blood subsidence
Abstract
A test value corresponding to blood subsidence from a
syllectogram is determined as a result of the effect of shearing
forces on a blood sample, by ascertaining the slope of the test
curve representing the syllectogram at a predetermined time
beginning with or after the onset of the blood aggregation phase as
the test value after: (a) sudden stoppage of the shearing; or (b) a
transition to a continuous minor residual shearing.
Inventors: |
Schmid-Schonbein; Holger
(Aachen, DE), Kiesewetter; Holger (Aachen,
DE), Mussler; Klaus (Aachen, DE), Myrenne;
Heinz (Roetgen, DE), Scheffler; Andreas
(Wuppertal, DE) |
Assignee: |
Ernst Leitz Wetzlar GmbH
(Wetzlar, DE)
|
Family
ID: |
25781585 |
Appl.
No.: |
06/141,638 |
Filed: |
April 18, 1980 |
Foreign Application Priority Data
|
|
|
|
|
Oct 20, 1979 [DE] |
|
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2942466 |
Mar 11, 1980 [DE] |
|
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3009260 |
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Current U.S.
Class: |
356/39; 356/40;
356/427 |
Current CPC
Class: |
G01N
15/05 (20130101) |
Current International
Class: |
G01N
15/04 (20060101); G01N 15/05 (20060101); G01N
033/48 () |
Field of
Search: |
;356/39,40,427
;250/556 |
References Cited
[Referenced By]
U.S. Patent Documents
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3706877 |
December 1972 |
Clifford, Jr. et al. |
4135819 |
January 1979 |
Schmid-Schonbein |
4252536 |
February 1981 |
Kishimoto et al. |
|
Other References
Quantitative Evaluation of the Rate of Rouleaux Formation of
Erythrocytes by Measuring Light Reflection ("Syllectometry"),
Brinkman et al., Reprinted From Proceedings Series C, 66, No. 3,
1963. .
"Microheology & Protein Chemistry of Pathological Red Cell
Aggregation (Blood Sludge) Studied in Vitro", Schmid-Schonbein et
al., Biorhcology, 1973, vol. 10, pp. 213-227. .
Brinkman, Zijlstra & Jansonius, "Syllectometry", Nov. 3, 1963,
Koninkl. Nederl. Akademie, C. 66, pp. 236-248. .
Schmid-Schonbein et al., "Rheoscope Chamber", Microvascular
Research, vol. 6, pp. 366-376, (1973). .
Schmid-Schonbein et al., "New Hemorheological Techniques etc.",
Recent Advances in Cardiovascular Disease, vol. II, Supp. Aug.
1981, pp. 27-39..
|
Primary Examiner: McGraw; Vincent P.
Attorney, Agent or Firm: Wells & Wells
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
Applicants claim priority under 35 USC 119 for application Ser. No.
P 29 42 466. 6, filed Oct. 20, 1979 and for Ser. No. 30 09 260.5
filed Mar. 11, 1980 in the Patent Office of the Federal Republic of
Germany.
Claims
We claim:
1. In a method for obtaining measured test values from blood
sedimentation corresponding to blood subsidence, by inserting a
blood specimen in a transparent measuring chamber, removing
erythrocyte aggregation present in the specimen, illuminating the
specimen with light, and measuring the amount of light leaving the
specimen over a given time, wherein:
(a) said specimen is inserted in a measuring chamber having upper
(11) and lower (10) means movable relative to one another and the
specimen comes in contact with said upper and lower means;
(b) erythrocyte aggregation present in said operation is
counteracted by movement of said upper means relative to said lower
means; and
(c) the amount of light leaving said specimen during the
aggregation process setting in again is photometrically measured
after sudden stopping or after transition to a continuous minor
residual shearing movement of said upper and lower means, and
photometric signals are generated; the improvement comprising:
(d) ascertaining a syllectogram from said generated photometric
signals;
(e) defining as the onset of the aggregation phase an extreme value
of the syllectogram within a predetermined time interval of about 5
seconds following said sudden stopping of shearing or transition to
a residual continuous shearing; and
(f) ascertaining said test values at a predetermined time within
approximately 2.5 seconds after onset of the blood aggregation from
a slope of a test curve representing said syllectogram.
2. The method of claim 1, wherein said continuous minor residual
shearing takes place in impulsive manner.
3. The method of claim 1, wherein said transition to continuous,
minor residual shearing takes place gradually.
4. The method of claim 1, wherein a test curve representing the
syllectogram is digitized.
5. The method of claim 4, wherein said digitizing takes place at a
frequency of 20 hertz.
6. The method of claim 4, wherein the digitized test values are
multiplied by a factor normalizing the incident light
intensity.
7. The method of claim 4, wherein the digitized syllectogram is
normalized within the predetermined testing time interval by
subtracting the test value defining the onset of the aggregation
phase.
8. The method of claim 7, wherein an area under the normalized,
digitized syllectogram is determined by numerical integration
during the predetermined time interval.
9. The method of claim 1, wherein testing takes place alternatively
after suddenly stopping shearing and after transition to residual
shearing.
10. The method of claim 1, wherein the step (f), said test values
are ascertained from an average slope within a time interval of
approximately 2.5 seconds after onset of the aggregation phase.
11. The method of claim 1, wherein in step (f), said test values
are ascertained from an area under a test curve representing said
syllectogram within a time interval of approximately 10 seconds
beginning with the onset of the aggregation phase.
12. In an apparatus for rapidly ascertaining measured test values
disclosing blood subsidence from a minimum amount of blood by
measuring the natural aggregation rate of particles in liquid
blood, comprising:
(a) means for illuminating and defining an optical axis;
(b) upper (11) and lower (10) means defining a transparent
anti-aggregation chamber therebetween for being filled with a
minimum amount of blood specimen, said blood specimen touching said
upper and lower means and located along said optical axis;
(c) drive means for agitating said anti-aggregation chamber by way
of slow motion of said upper means relative to said lower means and
for separating said blood specimen;
(d) rapid shut-off means to retard or to stop said agitated
chamber;
(e) photoelectric receiver means responsive to illumination leaving
said anti-aggregation chamber; and
(f) means for deriving electrical signals from said photoelectric
receiver means after retarding or stopping said chamber over the
whole aggregation time; the improvement comprising:
(g) an analyzing stage for providing a syllectogram from said
electric signals and defining as the onset of the aggregation phase
an extreme value of the syllectogram within a predetermined time
interval of about 5 seconds following said sudden stopping of
shearing or transition to a residual continuous shearing and
ascertaining said test values at a predetermined time beginning
within approximately 2.5 seconds after onset of blood aggregation
from a slope of a test curve representing said syllectogram.
Description
BACKGROUND OF THE INVENTION
The field of the invention is blood analysis by optics, measuring
and testing and the invention is particularly related to a method
and apparatus for determining a test value corresponding to blood
subsidence from a syllectogram of a blood sample that is being
subjected to shear forces.
The state of the art of determining a test value corresponding to
blood subsidence from a syllectogram of a blood sample that was or
is being subjected to shear forces may be ascertained by reference
to the article entitled "Syllectometry" by Zijlstra, as published
in the Proc. Koninkl. Nederl. Akad. v. Wetensch., Amsterdam, Ser.
C., Vol. 66, no. 3 (1963) at pp. 237-248.; Microvascular Research,
Vol. 6, (1973), pp. 366-376; and U.S. Pat. No. 4,135,819, of
Schmid-Schonbein, the disclosures of which are incorporated
herein.
U.S. Pat. No. 4,135,819 discloses an apparatus and method for
measuring the aggregation rate of capillary native blood free of
coagulation inhibitor in order to quickly ascertain information
disclosing blood subsidence from a minimum amount of blood. In U.S.
Pat. No. 4,135,819, the light transmission of a blood sample is
recorded as a function of time. This magnitude changes as a
function of the significant rheological phenomenon, the erythrocyte
aggregation, and therefore permits data to be obtained. Photometric
aggregometry may be measured both in transmission and reflection.
The test curve obtained was first coined "syllectogram" by
Zijlstra, op. cit. It is based on the fact that the scattering of
light in a blood sample decreases after aggregates are formed and
the transmission of light increases accordingly.
Three basic phases must be distinguished in the course of a
measurement, namely: (1) the mixing phase, (2) the stopping phase,
i.e., the phase of slight shearing, and (3) the aggregation phase.
To carry out the measurement, a blood sample is introduced into a
measuring chamber consisting essentially of a transparent,
disk-cone system rotating in the same or mutually opposite
directions. During the mixing phase, the erythrocytes orient
themselves under the influence of shearing forces and thus create
clear plasma spaces by means of which the light can pass through
the blood sample. In view of the material inhomogeneities in the
path of the light beam, the light transmission fluctuates about a
mean value.
When the shearing is terminated by abruptly stopping the disk-cone
system, there is an impulsive disorientation of the blood cells and
as a consequence of the elimination of clear plasma spaces, there
is also a reduction in the transmission.
With the ensuing onset of aggregation, the number of plasma gaps
grows again and therefore the transmission increases again. The
change of this transmission with time is essentially
exponential.
This time curve is quite reproducible, so that first the half-value
time characteristic of an exponential function was determined to be
the numerical value. It has been found, however, that no
unambiguous correlation could be obtained between this numerical
value and the conventionally obtained blood subsidence values.
It is furthermore known from Microvascular Research, op. cit., that
the syllectogram representing the time function of erythrocyte
aggregation presents a significantly higher test value in the
presence of a slight residual shearing than for the measurement at
rest.
U.S. Pat. No. 4,135,819 proposed not to use the measured curve, but
its derivative with time. This differentiated curve is also
exponential, whereby the erythrocyte aggregation again can be
represented by the half-value time of the differentiated
syllectogram. The half-value time is plotted by hand, or for a
rapid aggregation by electronic differentiation. But it has been
found that while the analog differentiation is quite suitable for
all pathological, i.e., rapid aggregation processes, it requires a
high expenditure in material for the slow ones. This is especially
the case for extremely decelerated aggregation which occurs in
healthy blood after it is diluted by an anticoagulant dissolved
therein.
SUMMARY OF THE INVENTION
Having in mind the limitations of the prior art, it is an object of
the present invention to analyze the syllectogram in a manner
independent of the function of time of the erythrocyte aggregation
and by a quick and simple way of measuring which can be visually
displayed as regards the rate of blood subsidence which is being
sought.
This object is achieved according to the present invention either
after a sudden stoppage of shearing or after a transition to a
continuous minor residual shearing. A test value is taken which is
the slope of the syllectogram at a predetermined instant after the
onset of the aggregation phase. This time is appropriately set at
2.5 seconds after onset of the aggregation phase. To decrease the
influence of accidental interference, advantageously the test value
is the mean slope of the syllectogram rather than a single slope
within a predetermined time interval from onset of the aggregation
phase. Again, this time interval selected is 2.5 seconds.
An alternative method is to determine the area under the
syllectogram within a predetermined time interval beginning with
the onset of the aggregation phase as the test value. This time
interval amounts to about 10 seconds. The onset of the aggregation
phase is determined by ascertaining an extreme value of the
syllectogram within a predetermined time interval following
stopping of the shearing or transition to a continuous minor
residual shearing. This time interval is selected to be 5
seconds.
The transition to a continuous minor residual shearing takes place
in mathematically continuous or discontinuous manner.
BRIEF DESCRIPTION OF THE DRAWINGS
The apparatus used in the present invention may best be explained
by reference to the appended drawings, wherein:
FIG. 1 is a plan view of an embodiment of the apparatus used in the
present invention shown in diagrammatic form;
FIGS. 1a and 1b show other embodiments for the mixing tub;
FIG. 2 is a detailed showing of an embodiment for the photoelectric
system 18 of FIG. 1;
FIG. 3 is a detailed showing of an embodiment for the analyzing
stage 19 of FIG. 1;
FIG. 4 is a plot showing a test curve representing a syllectogram
after sudden stoppage of the shearing or after a transition to a
continuous minor residual shearing; and
FIG. 5 is a plot showing the slope of a test curve representing a
syllectogram and the area under the test curve.
DESCRIPTION OF THE DRAWINGS
As shown in FIG. 1, the apparatus comprises a mixing chamber in the
design of a tub 10 receiving the blood to be tested. A cone 11
pivotable about its axis of rotation causing the mixing process
penetrates this tub. The tub and cone are made of a transparent
material and so dimensioned that for a given range of rotational
speeds of the cone, no significant centrifugal forces are applied
to the blood being tested. A drive 12 equipped with fast shut-off
actuates cone 11 which thus disperses the blood particles. Tub and
cone are illuminated from a light source 13 by means of a
deflecting mirror 13a and a condenser 14. Visual observation of the
test substance is made possible by an objective 15 and a splitter
17 and an ocular 16. Splitter 17 is followed by a photoelectric
system 18 which shapes as shown in FIG. 2 the light flux that comes
from the tub, cone, objective and splitter into proportional
electric signals. An analyzing stage 19 follows this system 18,
ascertaining the modification of the output signals from equipment
18.
In addition to the above description, optical components (reference
numerals 25, 26) are inserted along the optical axis of FIG. 1.
These components on account of their special design affect the
light amplitude alone of both the light amplitude and phase. As
shown, the components in the given embodiment consist of a stop 25
with circular aperture 27 positioned in front of chamber 10, and of
an annular structure located in a conjugate plane and adapted in
its dimensions to the image of annular stop 25. When this structure
for instance is a dyed-in layer, then both the amplitude and the
phase of the light reaching detector 18 will be affected. When
structure 26 is a neutral density filter, only the light amplitude
will be affected. The two measurements are appropriate when the
object to be examined predominantly affects the phase, but not the
amplitude of the light. The above offers but one of many possible
embodiments. Thus, tub and rotational body might also be made
spherical. Again, the mixing chamber itself may be used to disperse
the substance by being supported elastically and being shaken for
instance by one or several piezo electric resonators or
oscillators, or by a loud speaker. In such cases the chamber
appropriately will be of tubular form (FIG. 1a). Again, one may
disperse the blood inside a tubular mixing chamber by the to and
fro motion of a stirrer dipping into the tube and the motion of
which may be stopped abruptly (FIG. 1b).
When the blood being tested is put into motion by rotating the
cone, existing particle aggregates dissolve. But they reform when
cone rotation is stopped or retarded and hence the blood returns to
rest. This causes a change in transparency and hence a change in
the output signals from equipment 18. Test values may be displayed
digitally at stage 21 following stage 19, or they may be stored in
a memory 23. These components 21 and 23 are disclosed in U.S. Pat.
Nos. 3,317,736, FIG. 3 and 3,306,095, FIG. 1. When using a warning
system, the instrumentation shown may also be used for series
(assembly line) tests.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As regards the further electronic processing of the syllectogram,
it is especially advantageous to digitize the test curve. Good
resolution is achieved if digitizing takes place at a frequency of
20 Hz.
To carry out control measurements, the digitized test values
appropriately are multiplied by a factor normalizing the incident
light intensity. Information about the absolute,
aggregation-determined change in light intensity is obtained when
the digitized syllectogram is normalized within the predetermined
testing time interval by subtracting the test value defining the
onset of the aggregation phase. In a preferred embodiment of the
present invention, the area under the normalized digitized
syllectogram is determined by numerical integration during the
predetermined time interval.
Improved control for interpreting the test results is obtained when
measurements are carried out alternatingly for sudden stoppage
after shearing and after transition to a residual shearing.
The present invention is advantageously useful also for
agglutination research if a minor residual shearing is maintained
during the time of measurement.
Contrary to the prior art analytical methods for the syllectogram,
which must take into account the curve over a substantial length of
time in order to determine the half-value time, the present
invention is restricted to the curve of the first few seconds. In
particular cases, the time of measurement is determined not by the
particular curve, but by empirical data independent therefrom.
The actual time of measurement, which includes the stopping phase
and the aggregation phase, is for instance 7.5 seconds for the
slope measurement and 15 seconds for the area measurement, so that
a very rapid analytical process is available for diagnostic
purposes.
The present invention is based on the results from comprehensive
mathematical research in the curves representing the syllectogram.
By iteratively adapting to the analog curve and computing a
compensating spline, it is found that the function of the change of
light intensity in the aggregation phase represents a natural
process only during the first seconds, this function being
superposed by others in the further course of the test run.
The determination of the slope of the syllectogram at a given
instant, or the mean slope for a predetermined time interval after
onset of the aggregation phase is a measure of the rate of
aggregation. The area under the test curve within a predetermined
time interval quantifies the magnitude of the aggregation that took
place. Both magnitudes measure aspects of the primary aggregation
process which is also responsible for the conventional measurement
of the rate of blood subsidence.
It is manifest therefore that the values found for the rate of
aggregation are interpreted in the same manner as for the rate of
blood subsidence. But the values for the area also provide
numerical data proportional to those of the rate of blood
subsidence, as the area is zero when no aggregation takes place
after the stopping phase and a maximum when the aggregation is
practically fully completed within the time of testing. The area is
considerably smaller for healthy blood than for pathological blood,
and this agrees with the rate of blood subsidence in the normalized
range for wholly healthy individuals.
The variation in the above described method, wherein the stopping
phase ensuing the mixing phase is replaced by a phase of minor
shearing which is furthermore maintained during the following phase
of aggregation, is advantageous for instance when the test chamber
consists of a stator and a rotor for the disaggregation of the
erythrocytes. Depending on the thickness of the sample layer, a
flow gradient may develop on account of inertia within the sample
after the sudden stopping of the rotor, whereby time-decreasing
shearing forces are generated during the photometric test.
It is known from the earlier measurements conducted according to
the prior art cited above that a minor shearing action affects the
course of the (otherwise) natural erythrocyte aggregation. This
effect is more pronounced in pathological blood samples than in
healthy ones and is opposite to the latter. A time-varying
disaggregation determined by the flow conditions therefore affects
the natural aggregation in a manner which cannot be accounted for
by measurement techniques.
The transition of the present invention to a continuous minor
residual shearing on the other hand creates specific testing
conditions because the proportion of the shear-induced aggregation
then is a constant.
Advantageously also the measurements are carried out alternatingly
following the stopping phase and in the shear-induced phase, as the
comparison of the test results provides a further index for the
presence of pathological blood samples. The effect of the
shear-induced aggregation for instance is especially significant in
rheumatic sufferers.
New research furthermore has shown that the agglutination of blood
takes place at substantially accelerated pace when under the
influence of minor shearing forces. The known cross-match tests for
determining blood group incompatibility require a waiting time of
about half an hour when the conventional methods are used.
On the other hand, when a syllectogram is taken in the presence of
sustained shearing force, an indication is already provided after
about 30 seconds. This makes it possible for the first time to
ascertain blood group incompatibility during a blood transfusion
and to stop the transfusion from proceeding.
BEST MODE OF CARRYING OUT THE INVENTION
An illustration of the present invention using area determination
is described below in relation to a transmission syllectogram. To
record the syllectogram, the photometric system described in FIGS.
1 to 3 and U.S. Pat. No. 4,135,819 is used. A micro-computer with
suitable program modules is used to carry out the novel analytical
procedure.
The incident light intensity must be known before a test run begins
in order to carry out transmission measurements. To that end the
digital input of a digital-analog converter (DAC) is increased
until the measured value for the empty chamber is balanced to 100%
transmission.
The reference value for the incident intensity is represented by
the last value of the DAC input register and stored in the
microcomputer memory.
Thereupon the test chamber is filled with the blood sample. The
program starts the chamber drive to eliminate the aggregation which
is present. A transmission constantly fluctuating about a mean
value sets in. The end of the mixing phase is controlled in terms
of a given number of revolutions of the cone-disk system of the
test chamber. The end of the mixing phase is determined by a
revolution counter. The chamber drive motor is stopped and the test
data recording begins. The transmission data are transferred at a
frequency of 20 Hz for 15 seconds following motor stoppage by an
analog-digital converter (ADC) and stored in the micro-computer
memory. The reference value obtained for the incident light
intensity is kept constant through a testing cycle, and the
transmission data are referred to it.
Analysis begins with the determination of the start
(T.sub.o,t.sub.o) of aggregation. This is defined as the absolute
minimum of the transmission curve .pi.(t) within the first 5
seconds after motor stoppage.
By subtracting the transmission T.sub.o measured at the onset of
the aggregation from all subsequent measured values in the time
interval (t.sub.o,t.sub.o +10), the curve of the absolute changes
in transmission, referred to the initial value, caused by the
progressive aggregation of the erythrocytes, is obtained.
The transmission changes so computed can be used in a so-called
quadrant correlation test by Quenouille to determine whether
measurable aggregation took place at all in the tested blood
sample, because, as already mentioned, the aggregation may be null
in healthy subjects. If there is no aggregation, there is
consequently no integration of changes in transmission, rather the
result is indicated immediately.
The numerical integration of the computed changes in transmission
can be implemented by successively summing segments of area by
means of an algorithm fitted to the test procedure. Advantageously,
a compensating parabola is computed by means of 5 particular
successive measuring points with subscripts i.sub.n -i.sub.n+4
corresponding to a time interval of 0.2 seconds, this parabola
thereupon being integrated between the 3 central points i.sub.n+1
-i.sub.n+3. The set of values so obtained results from displacing
the computing templet by 2 increments in the direction of the
positive time axis. The new subscripts so obtained then are
i.sub.n+2 -i.sub.n+6.
As the sole exception, the beginning segment is integrated within
the first four points. This integration procedure ensures extreme
accuracy by the overlapping junction of the compensating parabolae
to the particular previous test points.
The total obtained can be displayed directly, but it can also be
multipled by a factor taking into account special influences
ascertained in particular patients as the normalized values. These
normalized values for instance may be related to age, sex,
hematocrit and similar values.
In lieu of the above described integration of area segments, the
slope of a straight line passing through two or more test points
obviously is obtained in numerical manner. The required
mathematical algorithms for that purpose are known and again can be
implemented by a microcomputer.
Furthermore, analog methods are known for ascertaining the slope of
a curve and the area under the curve.
* * * * *